Microorganism producing hyaluronic acid and purification method of hyaluronic acid

ABSTRACT

The present invention relates to a hyaluronic acid producing strain  Streptococcus  sp. KL0188 and a method for purifying hyaluronic acid, more particularly to a  Streptococcus  sp. KL0188 that does not express hyaluronidase and is non-hemolytic, and a method for purifying hyaluronic acid using an aromatic adsorption resin and an active carbon.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a hyaluronic acid producingmicroorganism strain and a method for purifying hyaluronic acid, andparticularly to Streptococcus sp. KL0188 and a method for purifyinghyaluronic acid using an aromatic adsorption resin and an active carbon.

(b) Description of the Related Art

Hyaluronic acid, which is a colorless highly viscous polysaccharidehaving a molecular weight of 50,000 to 13,000,000 Da, has repeat unitsof glucuronic acid and N-acetyl glucosamine that are alternately bondedas (1-3) and (1-4). Hyaluronic acid has a moisturizing effect, anexcellent lubricating effect against physical friction, and it offersexcellent protection effects against invasion of bacteria, etc.Hyaluronic acid is widely used as a cosmetic additive, a treating agentfor arthritis, a supplementary agent for ophthalmic operations, anadhesion inhibitor after surgical operations, etc. A lot of hyaluronicacid is contained in cow eyeballs, cock combs, buffer tissue of animals,placentas, cancer cells, skin, etc.

Hyaluronic acid can be extracted from the above-mentioned bio-tissues(U.S. Pat. No. 4,141,973 and U.S. Pat. No. 4,303,676), or it can becollected as a fermentation product by fermenting microorganisms.However, hyaluronic acid obtained by extraction contains impurities suchas chondroitin sulfate, glycosamino glycansulfate, etc. hence acomplicated purification process is required in order to remove theseimpurities causing a high production cost. However, according to theproduction method of hyaluronic acid using microorganisms, productioncost is comparatively low, and high molecular weight hyaluronic acid canbe obtained by a relatively simple method (Japanese Patent Laid-Openpublication No. 58-056692, U.S. Pat. No. 8,600,066).

Microorganisms used for the production of hyaluronic acid includeStreptococcus pyogenes, Streptococcus faecalis, Streptococcusdysgalactiae, Streptococcus zooepidemicus, Streptococcus equi,Streptococcus equisimilis, etc. According to Bergy's manual, thesepertain to Lancefield's serological group A or C. Such microorganismsare hemolytic Streptococcus, and they are reported to havebeta-hemolytic functions.

Since hyaluronic acids that are produced using Streptococcus sp.microorganisms (Japanese Patent Laid-Open Publication No. 58-566922,U.S. Patent Laid-Open Publication No. 60-500997, Korean PatentRegistration Publication No. 10-250573, and Korean Patent Laid-OpenPublication No. 10-250573) have relatively low average molecular weightsof 300,000 to 3,500,000 Da, it is difficult to use them as a medicinaltreating agent or supplementary agent, and they have insufficientmoisturizing power for cosmetics. In addition, U.S. Pat. No. 6,090,596describes a method for producing high molecular weight hyaluronic acidwith molecular weight of 6,300,000 to 9,500,000 Da, but productivity ofhyaluronic acid is 0.35 g per L of culture solution, which is very low.

Known methods for separating and purifying hyaluronic acid usingmicroorganisms are as follows:

U.S. Pat. No. 4,157,296 discloses a method for purifying 5 hyaluronicacid by treating a culture solution of Streptococcus pyogenes withtrichloro acetic acid to remove strains, and then precipitating it usingan organic solvent. However, since the precipitation method using anorganic solvent requires numerous repetitive operations, it has acomparatively high cost and consumes substantial time.

U.S. Pat. No. 4,782,046 describes a purification process of introducing0.01% anionic surfactant of lauryl sulfate into a culture solution ofStreptococcus equi to separate hyaluronic acid attached to cell walls,and then introducing a non-ionic surfactant of hexadecyltrimethylammonium bromide to form a hyaluronic acid precipitate, andprecipitating it with alcohol.

U.S. Pat. No. 4,784,990 describes a purification process of addingethanol to a culture solution of Streptococcus zooepidemicus to separatehyaluronic acid from microorganisms, and then precipitating it withcetyl pyridinium chloride.

Japanese Patent Laid-Open Publication No. 63-012293 describes a methodfor removing both low molecular weight hyaluronic acid with a molecularweight of 1,500,000 Da or less and exothermic material by treating ahyaluronic acid-containing solution with a macroreticular anion exchangeresin (Dianion HPA-25, HPA-75, IRA-900, IRA-904).

Japanese Patent Laid-Open Publication No. 13-131503 describes a methodfor purifying hyaluronic acid by treating a hyaluronic acid-containingsolution with alumina or silica gel, etc. to remove exothermic material,proteins, nucleic acid, metal impurities, etc., and precipitating itwith an organic solvent.

Japanese Patent Laid-Open Publication No. 06-199656 describes a methodfor purifying hyaluronic acid by passing a hyaluronic acid-containingsolution through a membrane filter charged on a solution of pH 6 to 10to remove exothermic material, and precipitating it with alcohol.

Korean Patent Laid-Open Publication No. 1994-2478 describes a method forpurifying hyaluronic acid by adding iron aluminate powders to ahyaluronic acid producing strain culture solution.

In addition, Korean Patent Laid-Open Publication No. 1997-42603describes a method for purifying hyaluronic acid by treating ahyaluronic acid-containing solution with a hydrophobic polymer(polyethylene, polypropylene, or polystyrene), and then adding activealumina, and precipitating it with alcohol.

However, the above-mentioned methods involve complicated treatingprocesses, which increase production cost, and it is difficult tocompletely remove exothermic material, proteins, nucleic acid, etc.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a microorganismstrain that can produce high molecular weight hyaluronic acid with ahigh yield.

It is another object of the present invention to provide a hyaluronicacid producing microorganism strain that does not express hyaluronidaseand is not hemolytic.

It is another object of the present invention to provide a highmolecular weight hyaluronic acid that is produced from a non-hemolyticmicroorganism strain and purified.

It is another object of the present invention to provide a method forpurifying hyaluronic acid produced from microorganisms, which can removeexothermic material in an easy and straightforward manner, and separatehyaluronic acid with high purity.

In order to achieve these objects, the present invention providesStreptococcus sp. KL0188 (KCTC1024BP), which does not expresshyaluronidase and is non-hemolytic.

The present invention also provides a method for purifying hyaluronicacid and a salt thereof, by treating a culture solution of a hyaluronicacid producing microorganism strain with an aromatic adsorption resin,treating it with an active carbon, and precipitating it with an organicsolvent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a hyaluronic acid producingmicroorganism strain and a purification method of hyaluronic acid.

According to the present invention, Streptococcus sp. KL0188 that isprepared by causing mutation in Streptococcus zooepidemicus is provided.The Streptococcus sp. KL0188 has been deposited with the KoreanCollection for Type Culture, on May 10, 2002, under deposit No.KCTC10248BP. The Streptococcus sp. KL0188 is a non-hemolytic strain, andit can produce hyaluronic acid with a high yield because it does nothave hyaluronidase activity.

The Streptococcus sp. KL0188 can be cultured on a culture mediumcontaining trace elements such as a carbon source, a nitrogen source,inorganic salts, vitamins, etc. As the carbon source, glucose, sucrose,galactose, or fructose can be used, and preferably glucose is used. Asthe nitrogen source, ammonium nitrate, ammonium sulfate, tryptone,peptone, yeast extract, or casamino acid can be used; and as theinorganic salt, sodium chloride, sodium phosphate, disodium phosphate,ferrous sulfate, or magnesium sulfate can be used.

The example of the culture medium for Streptococcus sp. KL0188 that isused in the present invention comprises: 20 to 80 g/L of glucose, 5 g/Lof yeast extract, 17 g/L of casein peptone, 7 g/L of glutamic acid, 0.7g/L of magnesium sulfate, 2.5 g/L of potassium phosphate, and 5.0 g/L ofsodium chloride.

The Streptococcus sp. KL0188 can be cultured at 30 to 37° C. underaerobic conditions. The pH of the culture solution is preferablymaintained within the range of 6.5 to 7.5, and as the pH changes duringculture it is preferably controlled by artificial means. The pH can becontrolled using a 5N NaOH solution or 1N HCI solution. If the pH goesbeyond the above range, production and molecular weight of hyaluronicacid may be varied.

Hyaluronic acid produced from Streptococcus sp. KL0188 can be separatedand purified by common methods (J. Soc. Cosmet. Japan. 22, 35-42 (1988))or by the purification method of the present invention. TheStreptococcus sp. KL0188 produces approximately 6.0 to 7.5 g/L ofhyaluronic acid, with a high average molecular weight of 4,000,000 Da ormore.

Therefore, according to the present invention, the Streptococcus sp.KL0188 can produce hyaluronic acid with a low cost and high yield, andhyaluronic acid can also be purified by a relatively simple method. Inaddition, the hyaluronic acid produced therefrom can be used forcosmetics or a medicinal treating agent or supplementary agent.

The purification method of the present invention, which differs from theexisting hyaluronic acid precipitation method using surfactants,comprises the steps of active carbon treatment, aromatic adsorptionresin treatment, ultra-filtration, and ethanol precipitation of ahyaluronic acid producing microorganism strain culture solution.

As the aromatic adsorption resin, a styrene divinyl benzo-type resinfrom Mitsubishi Company can be used. Specifically, it is selected fromthe group consisting of HP10, HP20 (styrene and divinylbenzenecopolymer), HP21, HP30, SP800, SP825, SP850, SP875, SP205, SP206, andSP207 (brominated polystyrene), and preferably HP20 or SP207 is used.

As the hyaluronic acid producing strain, any strain that produceshyaluronic acid as a metabolite can be used, and representatively,Streptococcus sp. microorganisms can be used. The Streptococcus sp.microorganisms include Streptococcus pyogenes, Streptococcus faecalis,Streptococcus dysgalactiae, Streptococcus zooepidemicus, Streptococcusequi, Streptococcus equisimilis, and Streptococcus sp. KL0188(KCTC10248BP). The hyaluronic acid producing strains can be cultured bya common culture method to prepare a culture solution comprisinghyaluronic acid.

More specifically, the purification method of hyaluronic acid comprisesthe steps of (a) preparing a culture filtrate from a culture solution ofa hyaluronic acid producing strain; (b) adding an aromatic adsorptionresin to the culture filtrate, agitating, and conducting ultrafiltrationto prepare a hyaluronic acid solution; and (c) adding an organic solventto the hyaluronic acid aqueous solution to precipitate hyaluronic acidor a salt thereof, and drying it. The method further comprises the stepof adding active carbon to the culture filtrate or to the hyaluronicacid aqueous solution and agitating it, and then removing the activecarbon, after the step (a) or (b).

In the step (a), lauryl sulfate and formalin are added to the culturesolution and they are agitated, thereby separating hyaluronic acid fromthe bacteria surface and simultaneously deactivating the bacteria. Then,the culture solution is centrifuged to separate a supernatant, or it isfiltered to obtain a filtrate.

The step (b) is conducted after titrating the culture filtrate to a pHof 7.5 to 8.5, and the aromatic adsorption resin is added in an amountof 0.1 to 10 wt %. After adding the aromatic adsorption resin, the mixedfiltrate is agitated at 4 to 10° C. so that endotoxins are adsorbed, andit is then filtered to obtain a filtrate from which the adsorption resinis removed. Ultrafiltration of the filtrate is conducted to removevarious culture medium ingredients and inorganic salts. Theultrafiltration can be conducted using a filtration membrane with amolecular cut-off of 10,000 to 100,000 Da.

In the step (c), hyaluronic acid or a salt thereof is precipitated by acommon organic solvent precipitation method. As the organic solvent, anaqueous organic solvent such as acetone, methanol, ethanol, propanol,isopropanol, or acetonitrile can be used, and preferably ethanol isused. NaCl is added to a hyaluronic acid aqueous solution to theconcentration of 0.5 to 3M, the solution is filtered, and an organicsolvent is added to the filtrate in a volume of 1 to 5 times that of thefiltrate to precipitate hyaluronic acid and its salt. The precipitate isthen washed with 70% ethanol and dried.

The active carbon treatment can be conducted after the step (a) or (c).Specifically, to the culture filtrate of the step (a) or to thehyaluronic acid of the step (b), 0.1 to 3 (w/v)% of NaCl is added, andthen 0.1 to 10 (w/v) % of active carbon is added. The active carbon isused to adsorb proteins or nucleic acid to remove it.

The purification method of hyaluronic acid can efficiently removeexothermic material, proteins, nucleic acid, and metal impurities,compared to the conventional method, and can minimize organic solventprecipitation frequency and hence prepare hyaluronic acid with a highpurity by a simple and economical purification process. Accordingly,hyaluronic acid purified by the purification method of the presentinvention has a high purity of 99% and thus it can be used for cosmeticsor medicines.

The present invention will be described with reference to the followingexamples. However, these are only to illustrate the present inventionand the present invention is not limited to them.

EXAMPLE 1 Screening of Mutant Strain

Mutation was caused on Streptococcus zooepidemicus to select mutantstrains that have non-hemolytic properties and do not have hyaluronidaseactivities.

Streptococcus zooepidemicus (KCTC 3318) was inoculated on 50 ml of BacoTodd Hewitt Broth from DIFCO Company and cultured at 37° C. until analgebraic growth period occurred. Then, 1 ml of the culture solution wascentrifuged at a low temperature to recover precipitated cells, and 50mM of tris-maleic acid buffer solution (pH 6.0) was added thereto andwashed twice.

The cells were dispersed in a buffer solution at a concentration of1×10³ cells/ml, and NTG (N-methyl-N′-nitrosoguanidine) was mixedtherewith at a concentration of 200 μg/ml. The mixture was agitated at37° C. for 30 minutes, and then the cells were washed with 50 mM oftris-maleic acid buffer solution (pH 6.0) twice. The cells wereinoculated on Todd Hewitt Broth and cultured at 37° C. for 18 hours. Theculture solution was obtained and then diluted with sterile salinesolution to a concentration of 1×10³ cells/ml, and 0.1 ml of the dilutedsolution was cultured on Blood Agar to select colonies that did not showhemolysis.

On the selected non-hemolytic mutant strains, mutation was caused by thesame method as mentioned above to select strains that do not havehyaluronidase activity. The non-hemolytic mutant strains were coated ona Todd Hewitt Agar Broth containing 400 μg of hyaluronic acid and 1%albumin fraction V so that a single colony could be formed. Standingculture was conducted in a wet chamber at 37° C. for 2 to 5 days, andthen 10 ml of 2N acetate solution was added and stood for 10 minutes.Colonies that showed rapid growth and largely form viscous material wereselected.

The selected colonies were respectively inoculated on 1.5 L of culturemedium for hyaluronic acid production, and aerobically cultured at 35°C., pH 6.95 to 7.05, for 20 hours. The culture solution was recoveredand absolute viscosity was measured at 25° C. using a digital viscometer(Brookfield DVII+, 4 spin, 30 rpm). A part of the solution wasprecipitated with an organic solvent, dissolved in distilled water, andhyaluronic acid production was quantified by the carbazole method (Z.Dische, J. Biol. Chem. 167, 189 (1949)), thereby selecting strains thathave high absolute viscosity of culture solution and show highhyaluronic acid production.

The selected strains were identified by analyzing the amino acidsequence of 16S rDNA (Jukes, T. H. & C. R., (1969). In mammalian proteinmetabolism, pp. 21-132; Edited by H. N. Munro., Saito, N. & Nei, M.(1987) Mol Biol vol 4, 406-425). As a result, the selected strains wereidentified as Streptococcus sp. hence they were named Streptococcus sp.KL0188. The Streptococcus sp. KL0188 was deposited with the KoreanCollection for Type Culture on May 10, 2002, under deposition No. KCTC10248BP.

EXAMPLE 2 Inspection of Hyaluronic Acid Productivity

Streptococcus sp. KL0188 was cultured to measure hyaluronic acidproduction efficiency and the molecular weight of produced hyaluronicacid.

The separated microorganisms were inoculated on 100 ml of Todd HewittBroth and cultured at 35° C. until an algebraic growth period occurred,and then used as a first seed culture solution. The first seed culturesolution was inoculated on 1 L of tryptic soy broth (Difco, USA) andcultured at 35° C. until an algebraic growth period occurred, and thenused as a second seed culture solution.

In a 30 L fermenter, a hyaluronic acid production culture mediumcontaining 60 g/L of glucose, 5 g/L of yeast extract, 17 g/L of caseinpeptone, 7 g/L of glutamic acid, 0.7 g/L of magnesium sulfate, 2.5 g/Lof dipotassium phosphate, and 5.0 g/L of sodium chloride was introducedand sterilized, and 1000 ml of the second seed culture solution wasinoculated therein. Aerobic culture was conducted while maintaining thepH of the culture solution within the range of 6.95 to 7.05, atemperature of 35° C., and a ventilation amount of 1.0 VVM.

During culture, some amount of the sample was taken and viscosity of theculture solution was measured, and culture was conducted until theviscosity no longer increased. It was determined through measuring thatthe viscosity of the culture solution did not increase after beingcultured for 20 hours, and thus the culture was stopped. Maximumviscosity was approximately 20,000 cps.

Hyaluronic acid existing in the culture solution was recovered by aknown separation and purification method (J. Soc. Cosmet. Japan. 22,35-42 (1988)). Hyaluronic acid was quantified by a carbazole method (Z.Dische, J. Biol. Chem. 167, 189 (1947)), and the average molecularweight of the produced hyaluronic acid was measured by a capillaryviscometer method (Narlin, Analytical Biochemistry 147, 347-395 (1985)).As a result, hyaluronic acid production amount was confirmed as 7.0 g/Land the average molecular weight was 5,500,000 Da.

COMPARATIVE EXAMPLE 1 Examination of Hyaluronic Acid Productivity ofStreptococcus zooepidemicus

Streptococcus zooepidemicus (KCTC3318) was cultured by the same methodas in Example 2, and hyaluronic acid productivity and molecular weightwere measured.

After 24 hours, viscosity of the culture solution did not increase andthus culture was stopped. The viscosity was measured to be approximately4000 cps, hyaluronic acid productivity was 3.0 g/L, and averagemolecular weight was 2,500,000 Da.

It was confirmed that the Streptococcus sp. KL0188 of the presentinvention has excellent hyaluronic acid productivity and the molecularweight of the produced hyaluronic acid was high, compared toStreptococcus zooepidemicus.

The Streptococcus sp. KL0188 of the present invention is a non-hemolyticstrain, and produces hyaluronic acid with a high molecular weight and ahigh yield. Therefore, hyaluronic acid produced from the

Streptococcus sp. KL0188 can be used for cosmetics or medicines.

EXAMPLE 3 Purification of Hyaluronic Acid Using Aromatic AdsorptionResin SP207

3-1. Preparation of Hyaluronic Acid Producing Strain

Streptococcus sp. KL0188 (KCTC10248BP) was inoculated on 100 ml of ToddHewitt Broth and cultured at 35° C. until an algebraic growth periodoccurred, and then used as a first seed culture solution. The firstculture solution was inoculated on 1 L of tryptic soy broth (Difco, USA)and cultured at 35° C. until an algebraic growth period occurred, andthen used as a second seed culture solution.

To a 30 L fermenter, 20 L of hyaluronic acid production culture mediumcontaining 60 g/L of glucose, 5 g/L of yeast extract, 17 g/L of caseinpeptone, 7 g/L of glutamic acid, 0.7 g/L of magnesium sulfate, 2.5 g/Lof potassium diphosphate, and 5.0 g/L of sodium chloride was introducedand sterilized, and 1000 ml of the second seed culture solution wasinoculated thereon. Culture was conducted at a temperature of 35° C. anda pH of 6.95 to 7.05 for 20 hours.

3-2. Preparation of Culture Filtrate

The hyaluronic acid culture solution was diluted such that theconcentration of hyaluronic acid became 0.1 to 0.2%. 0.02% laurylsulfate and 0.05% formalin solution were added thereto and agitated for3 hours. Then, bacteria were removed by centrifugation or filtration toprepare a culture filtrate.

3-3. SP207 Resin Treatment

The culture filtrate was titrated such that the pH became 7.5 to 8.5, 3(w/v) % of aromatic adsorption resin SP207 was added, and exothermicmaterial was adsorbed while agitating at 4 to 10° C. for 3 hours. Then,the adsorption resin-treated solution was filtered to obtain a filtratefrom which the adsorption resin was removed, and ultrafiltration wasconducted.

3-4. Active Carbon Treatment

To the filtrate, 0.9 (w/v) % of NaCl and 3 (w/v) % of active carbon wasadded, and it was agitated for 2 hours to adsorb protein and nucleicacid, etc. into the active carbon. Then, filtration was conducted toobtain a hyaluronic acid aqueous solution from which the active carbonwas removed.

3-5. Ethanol Precipitation

To the hyaluronic acid aqueous solution, NaCl was added such that itsconcentration became 1 M, and the solution was filtered using a 0.2 μmfilter. Then, ethanol was added in a volume of 1.5 to 3 times that ofthe solution to precipitate hyaluronic acid and its salt, and it wasthen washed with 70% ethanol several times. The precipitate was driedunder sterilized conditions to obtain hyaluronic acid and its salt.

EXAMPLE 4 Purification of Hyaluronic Acid Using Aromatic AdsorptionResin HP20

Hyaluronic acid and its salt were purified by the same method as inExample 3, except that HP20 was used as an adsorption resin.

EXAMPLE 5 Purification of Hyaluronic Acid Using Aromatic AdsorptionResin SP207 after Active Carbon Treatment

Hyaluronic acid and its salt were purified by the same method as inExample 3, except that SP207 was adsorbed after the active carbontreatment.

EXAMPLE 6 Purification of Hyaluronic Acid Using Aromatic AdsorptionResin HP20 after Active Carbon Treatment

Hyaluronic acid and its salt were purified by the same method as inExample 4, except that HP20 was adsorbed after the active carbontreatment.

EXAMPLE 7 Purification of Hyaluronic Acid Using SP207

Hyaluronic acid and its salt were purified by the same method as inExample 3, except that Streptococcus zooepidemicus (KCTC3318) was usedas a hyaluronic acid producing strain.

COMPARATIVE EXAMPLE 2

Hyaluronic acid and its salt were purified by the same method as inExample 3, except that an aromatic resin adsorption step was omitted.

Experiment: Measurement of Hyaluronic Acid Purification Yield

The purities of the hyaluronic acid and its salts purified by themethods of Examples 3 to 7 and Comparative Example were measured.

A. Hyaluronic acid yield: Hyaluronic acid yield was quantified by amodified carbazole method, and initial volume and final volume werecompared.

B. Measurement of exothermic material: Hyaluronic acid and its salt weredissolved in water containing exothermic material of 0.001 EU/ml or lesssuch that its density became 1.5 g/L, and then analyzed using LAL(Limulus Amebocyte Lysate) from Charles River Endosafe according to theattached manual. The analyzed value was shown as endotoxin units (EU)existing per 1 mg of hyaluronic acid.

C. Purity test: The purity test was conducted by a carbazole method(Anal. Biochem., 4,330 (1962)).

D. Protein content: The protein content was measured by the Lowrymethod.

E. Nucleic acid content: Hyaluronic acid and its salt were dissolved ina saline solution to 1%, and then adsorbancy was measured at 260 nm.

The results were described in the following Table 1. TABLE 1 ResultsExample Example Example Example Example Comparative Item 3 4 5 6 7Example 2 Yield    78%    80%    82%    79%    78%  79% Purity(%) 99.099.0 99.0 99.0 99.0 95 Exothermic <0.005 <0.005 <0.005 <0.005 <0.005 0.1Material (EU/mg) Protein (%) <0.01% <0.01% <0.01% <0.01% <0.01% 0.2Nucleic acid ND ND ND ND ND 0.1% Molecular weight 5.3 5.2 5.5 5.4 2.55.4 (million Da) Metal ions Iron(ppm) <1 ppm <1 ppm <1 ppm <1 ppm <1 ppm<1 ppm Lead(ppm) <1 ppm <1 ppm <1 ppm <1 ppm <1 ppm <1 ppm arsenic <1ppm <1 ppm <1 ppm <1 ppm <1 ppm <1 ppm (ppm)

As can be seen from Table 1, the hyaluronic acid and its salt purifiedby Examples 1 to 7 had a high purity of 99%. Meanwhile, the hyaluronicacid purified by Comparative Example had a purity of 95%. In addition,the hyaluronic acid and its salt of Examples 3 to 7 have much lowercontents of exothermic material, protein, and nucleic acid thanComparative Example.

1. Streptococcus sp. KL0188 (KCTC), which is a hyaluronic acid producingmicroorganism strain that does not express hyaluronidase and that showsa non-hemolytic property.
 2. A method for purifying hyaluronic acid,comprising the steps of treating a culture solution of the Streptococcussp. KL0188 (KCTC10248BP) of claim 1 with an aromatic adsorption resin,treating it with an active carbon, and precipitating it with an organicsolvent to purify hyaluronic acid and a salt thereof.
 3. The method forpurifying hyaluronic acid according to claim 2, wherein the aromaticadsorption resin is selected from the group consisting of HP10, HP20,HP21, HP30, SP800, SP825, SP850, SP875, SP205, SP206, and SP207.
 4. Themethod for purifying hyaluronic acid according to claim 2, wherein themethod comprises the steps of: (a) preparing a culture filtrate from theculture solution of the hyaluronic acid producing microorganism strain;(b) adding an aromatic adsorption resin to the culture filtrate,agitating it, and conducting ultrafiltration to prepare a hyaluronicacid aqueous solution from which exothermic material is removed; and (c)adding an organic solvent to the hyaluronic acid aqueous solution toprecipitate hyaluronic acid and a salt thereof, and drying it, whereinthe method further comprises the step of adding an active carbon to theculture filtrate or to the hyaluronic acid aqueous solution and removingthe active carbon, after the step of (a) or (c).
 5. A hyaluronic acidand a salt thereof purified by the method of claim
 4. 6. A method forpurifying hyaluronic acid comprising the step of treating a culturesolution of hyaluronic acid producing strain with an aromatic adsorptionresin, treating it with an active carbon and precipitating it with anorganic solvent to purify hyaluronic acid and a salt thereof.
 7. Themethod for purifying hyaluronic acid and a salt thereof according toclaim 6, wherein the aromatic adsorption resin is a copolymer of styreneand divinylbenzene, or brominated polystyrene.
 8. The method forpurifying hyaluronic acid and a salt thereof according to claim 6,wherein the aromatic adsorption resin is selected from the groupconsisting of HP10, HL20, HP21, HP30, SP800, SP850, SP875, SP205, SP206,and SP207.
 9. The method for purifying hyaluronic acid and a saltthereof according to claim 6, wherein the method comprises the steps of:(a) preparing a culture filtrate from the culture solution of hyaluronicacid producing microorganism strain; (b) adding an aromatic adsorptionresin to the culture filtrate, agitating it, and conductingultrafiltration to prepare a hyaluronic acid aqueous solution from whichexothermic material is removed; and (c) adding an organic solvent to thehyaluronic acid aqueous solution to precipitate hyaluronic acid and asalt thereof, and drying it, wherein the method further comprises thestep of adding an active carbon to the culture filtrate or to thehyaluronic acid aqueous solution and removing the active carbon, afterthe step of (a) or (c).
 10. The method for purifying hyaluronic acid anda salt thereof according to claim 6, wherein the hyaluronic acidproducing microorganism strain is a Streptococcus sp. strain.